Complexes formed of chelating agents and suitable specific metals are already used as contrastographic agents in the following diagnostic techniques: X ray imaging, nuclear magnetic resonance imaging (M.R.I.) and scintigraphy.
In particular, medical diagnosis using "magnetic resonance imaging" (M.R.I.), recognized as a powerful diagnostic agent in clinical practice (Stark, D. D., Bradley, W. G. , Jr. , Eds. "Magnetic Resonance Imaging" The C. V. Mosby Company, St. Louis, Mo. (USA), 1988), employs, above all, Paramagnetic pharmaceutical compositions, preferably containing complex chelates of bi-trivalent paramagnetic metal ions with aminopolycarboxylic acids and/or their derivatives or analogues.
Some of them are at present in clinical use as contrast agents for M.R.I. (Gd-DTPA, N-methylglucamine salt of the gadolinium complex with diethylentriaminopentacetic acid, MAGNEVIST.RTM., Schering; Gd-DOTA, N-methylglucamine salt of the gadolinium/1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid complex, DOTAREM.RTM. Guerbet).
In order to illustrate the state of the art in this field, here follows a list, incomplete, though indicative, of significant patent documents: EP 71564 (Schering), U.S. Pat. No. 4,639,365 (Sherry), U.S. Pat. No. 4,615,879 (Runge), DE-A-3401052 (Schering), EP 130934 (Schering), EP 65728 (Nycomed), EP 230893 (Bracco), U.S. Pat. No. 4,826,673 (Mallinckrodt), U.S. Pat. No. 4,639,365 (Sherry), EP 299795 (Nycomed), EP 258616 (Salutar), WO 8905802 (Bracco).
The contrast agents listed above and on the market are designed for a completely general use. In fact, after administration the MRI contrast agent is distributed in the extracellular spaces in different parts of the body prior to being excreted. In this sense they behave in a similar manner to iodine compounds used in X ray medical diagnosis.
Today, more than ever, the medical profession is in need of contrast agents that are aimed at specific organs, a need which is not adequately met by the products on the market at present. Especially, there is a need for contrast agents for the liver, an organ which is particularly prone to tumoral metastasis and which are almost always carcinomatose metastasis. Agents of this type should be able to provide the following results:
a) to clearly and selectively show the healthy tissue of the liver, thereby permitting the pin-pointing of small lesions such as metastasis (focal liver disease); PA1 b) an indication of hepatic function, whereby a disease as widespread as cirrhosis of the liver may be clearly exposed; PA1 c) a high resolution visualization of the bile ducts and of the gall bladder. PA1 the molecule enters the hepatocite through the sinusoid membrane following a mechanism that may or may not be specific (mediated by a carrier or a receptor). PA1 inside the hepatocite the molecule may: 1) be carried unaltered and linked to an intracellular protein or inside a vesicle, 2) undergo a conjugation reaction with an enzyme and be excreted in the bile as a conjugate, 3) be enzymatically degraded inside the lisosomes. PA1 the molecule leaves the hepatocite through the bile canaliculus membrane via a mechanism mediated by a carrier or through an exocytosis mechanism (if the molecule is carried inside the vesicle). PA1 bile acid carriers PA1 a bilirubin carrier PA1 a fatty acid carrier PA1 a carrier for organic cations PA1 A is the residue of a bile acid, wherein by bile acid the group of the bile acids obtainable by bioconversion from cholesterol is meant, particularly the acids: cholic, deoxycholic, chenodeoxycholic, ursodeoxycholic, lithocholic, and the derivatives thereof, including those with taurine and glycine; PA1 L is a linker between one of the C-3, C-7, C-12 or C-24 positions of the residue of the bile acid and B, corresponding to a group of formula (II) ##STR1## in which m is an integer varying from 1 to 10, wherein for values above 1, PA1 Y can have different meanings, PA1 Y corresponds to the following succession of groups, ##STR2## n, l and q can be 0 or 1, p can vary from 0 to 10, PA1 X is an O atom, a S atom, or a --NR group, PA1 R is a H atom, or a (C.sub.1 -C.sub.5) alkyl group, PA1 K is benzene ring, substituted or not, or a --CHR.sub.1 group, PA1 R.sub.1 is an hydrogen atom, or a --COOH group, or a --SO.sub.3 H group, PA1 Z is an O atom or a S atom, or one of the --CO-- or --CS-- groups, PA1 B is the residue of a chelating agent of bi-trivalent metal ions having an atomic number varying from 20 to 31, 39, 42, 43, 44, 49, or from 57 to 83, wherein this residue can in its turn be conjugated or not, by a second chain L of formula (II), to another residue A as defined above, PA1 1) synthesis of a functionalized ligand i.e. of a ligand capable of coordinating one paramagnetic metal ion and at the same time of binding stably to the bile acid by means of a suitable functional group; PA1 2) synthesis of a functionalized bile acid; PA1 3) coupling reaction between two different syntons; PA1 4) cleavage of any protective groups; PA1 5) complexation of the paramagnetic metal ion.
Primary hepatic carcinoma (HCC) is a pathology which has become increasingly and rapidly widespread in the last twenty years, both in the Western World and in Japan (Okuda K., Hepatology, 15, 948,1992). As a result of this, the need for a fast and efficient method of diagnosis for the detection of HCC emerges; for this purpose, Magnetic Resonance takes on a leading role, the proviso being the availability of a contrast agent which allows for the differentiation between the healthy hepatocites and those which are affected.
Today, only one product (AMI-HS of Advanced Magnetics, Reimer, P.; Weissleder, R. et al.; Radiology 177, 729, 1990, patent application WO-9001295) seems to possess the necessary prerequisites for the diagnosis of HCC. One is dealing with "ultra-small" particles of iron oxide (average diameter: 12 nm) coated with arabinogalactose which have a particular affinity with the asialoglycoprotein receptors present on the surface of the hepatocites. However, the use of these particles brings about various side effects, especially with regard to the circulatory system. The identification of an ideal hepatospecific contrast agent is, therefore, still far off.
Among the M.R.I. contrast agents under development, both the compound known as Gd-BOPTA (BRACCO, EP 230893), and the Schering product Gd-EOB-DTPA (EP-A-405704) turned out to be particularly suitable for the visualization of hepatic tissue, due to their characteristics of also being excreted via the bile tract.
The transport of both endogenic and xenobiotic substances by means of the hepatocites and the biliary excretion mechanisms have been amply discussed in the literature, only a few basic concepts of which shall be recalled as follows, see, for example, Meier, P. J. in "Biliary Excretion of Drugs and Other Chemicals", Siegers, C. -P. and Watkins III J. B. Eds. Gustav Fischer Verlag, Stuttgart, 1991.
The passage of a molecule in bile from blood through the Disse space takes place in numerous stages that may be schematically summarized as follows:
If the object is to synthesize a hepatotropic contrast agent which enters the hepatocites, the mechanisms that turn out to be the most interesting are those which are mediated by a receptor or a carrier. Up to now, the following carriers have been identified and partially characterized on the membrane sinusoid:
The first two types of carrier have been studied more in depth and the knowledge with regard to them is far more advanced.
The HCC cellular lines studied to date turn out to be made up of hepatocites which possess the bilirubin carriers. As both Gd-BOPTA and Gd-EOB-DTPA seem to penetrate the interior of the hepatocites taking advantage of this carrier, both products may not be of any help in the diagnosis of HCC, because they are not capable of differentiating between healthy and affected hepatocites.
It has been shown that in some human HCC lines the hepatocites are free from taurcalcoholic acid carriers (yon Dippe, P; Levy, D.; J. Biol. Chem. 265, 5942, 1990 and cited references). It appears, therefore, that research for a contrast agent that utilizes this carrier for penetrating hepatocites is of great interest.
Patent Application (EP-A-279307, Abbott) claims polyaminocarboxylic chelant conjugates, able to complex metal ions, with different substrates, among which are the bile acids. The only illustrative complex in the case of this Patent Application is a .sup.111 In complex of a conjugate in which a functionalized derivative of EDTA is covalently linked, through an amide link, to the carboxylic function of cholic acid. The possibility of chelating paramagnetic metal ions for the use in MRI is not mentioned.
Another Patent Application (EP-A-417725, Hoechst) generally claims products in which a bile acid is conjugated with pharmacologically active residues such as peptids, antibiotics, antivirals, renin inhibitors and medicaments for the treatment of diabetes. Recently, the results of the use of bile acid conjugates with chlorambucil, an antitumoral agent with cytotoxic action are reported by Kramer, W. et al.; J. Biol. Chem, 267,18598, 1992.